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Raf-1 regulates Rho signaling and cell migration.

Ehrenreiter K, Piazzolla D, Velamoor V, Sobczak I, Small JV, Takeda J, Leung T, Baccarini M - J. Cell Biol. (2005)

Bottom Line: These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane.Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration.Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Genetics, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

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Keratinocyte-specific disruption of c-raf-1 by the Cre-loxP system. (A) PCR genotyping of keratinocytes (KC) and tail genomic DNA preparations (T). M, marker. (B) Lack of Raf-1 protein in c-raf-1Δ/Δep keratinocytes. Keratinocytes isolated from six independent animals were lysed and subjected to Western blotting with an α-Raf kinase domain mAb, which recognizes all three Raf kinases. The genotype of the mice is indicated at the bottom of the panel. The band corresponding to Raf-1 is absent in K5-Cre+ cultures. (C) c-raf-1Δ/Δep mice display waved fur during the first hair cycle. A 4-wk old f/f and a c-raf-1Δ/Δep littermate are shown. (D) Normal epidermal architecture in c-raf-1Δ/Δep newborn mice. Paraffin sections from f/f or c-raf-1Δ/Δep skin were stained with hematoxylin/eosin.
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fig1: Keratinocyte-specific disruption of c-raf-1 by the Cre-loxP system. (A) PCR genotyping of keratinocytes (KC) and tail genomic DNA preparations (T). M, marker. (B) Lack of Raf-1 protein in c-raf-1Δ/Δep keratinocytes. Keratinocytes isolated from six independent animals were lysed and subjected to Western blotting with an α-Raf kinase domain mAb, which recognizes all three Raf kinases. The genotype of the mice is indicated at the bottom of the panel. The band corresponding to Raf-1 is absent in K5-Cre+ cultures. (C) c-raf-1Δ/Δep mice display waved fur during the first hair cycle. A 4-wk old f/f and a c-raf-1Δ/Δep littermate are shown. (D) Normal epidermal architecture in c-raf-1Δ/Δep newborn mice. Paraffin sections from f/f or c-raf-1Δ/Δep skin were stained with hematoxylin/eosin.

Mentions: We have used a Cre transgene expressed in the basal epidermal layer and follicular keratinocytes (Tarutani et al., 1997), to induce epidermis-specific ablation of the c-raf-1 gene. Primary keratinocytes derived from mice carrying an homozygous c-raf-1flox/flox (f/f) allele (Jesenberger et al., 2001) and the K5Cre transgene (c-raf-1Δ/Δep, deleted in epidermis) showed complete conversion of the c-raf-1flox to the c-raf-1Δ allele by PCR and lacked Raf-1 protein (Fig. 1, A and B). c-raf-1Δ/Δep mice were viable, fertile, and healthy. At 4 wk old, they displayed curled whiskers and a wavy fur (Fig. 1 C), a phenotype lost after the first hair cycle. The architecture of the epidermis (Fig. 1 D) as well as the expression of basal keratin and differentiation markers in the epidermis and hair follicles was indistinguishable in c-raf-1Δ/Δep mice and control littermates (not depicted). Thus, Raf-1 is not essential in epidermal development and homeostasis. Raf-1 ablation, however, markedly affected wound healing. Control mice closed and reepithelialized full thickness wounds (6-mm ∅) by day 9 after wounding, whereas >40% of the wound was still open in c-raf-1Δ/Δep mice at this time (not depicted). The wounds were not yet closed on day 12, when control mice had already completed both reepithelialization and clearance of cell debris underneath the wound crust (Fig. 2 A). c-raf-1Δ/Δep mice healed the wounds 4–5 d later, and the healed skin was indistinguishable from that of f/f mice (not depicted). The secondary responses of dermal components (granulation, inflammation, and neo-vascularization) were not affected by Raf-1 ablation in keratinocytes. The number of proliferating keratinocytes around the wound site was similarly unaffected (31 ± 10.8 Ki67+ cells in f/f, 32.5 ± 8.8 in Δ/Δep), and TUNEL staining of the migrating epithelial sheets did not show any apoptotic cells in c-raf-1Δ/Δep or control wounds (not depicted). Thus, lack of Raf-1 significantly delayed wound healing in the absence of proliferation or survival defects, suggesting that Raf-1 might be needed for keratinocyte migration.


Raf-1 regulates Rho signaling and cell migration.

Ehrenreiter K, Piazzolla D, Velamoor V, Sobczak I, Small JV, Takeda J, Leung T, Baccarini M - J. Cell Biol. (2005)

Keratinocyte-specific disruption of c-raf-1 by the Cre-loxP system. (A) PCR genotyping of keratinocytes (KC) and tail genomic DNA preparations (T). M, marker. (B) Lack of Raf-1 protein in c-raf-1Δ/Δep keratinocytes. Keratinocytes isolated from six independent animals were lysed and subjected to Western blotting with an α-Raf kinase domain mAb, which recognizes all three Raf kinases. The genotype of the mice is indicated at the bottom of the panel. The band corresponding to Raf-1 is absent in K5-Cre+ cultures. (C) c-raf-1Δ/Δep mice display waved fur during the first hair cycle. A 4-wk old f/f and a c-raf-1Δ/Δep littermate are shown. (D) Normal epidermal architecture in c-raf-1Δ/Δep newborn mice. Paraffin sections from f/f or c-raf-1Δ/Δep skin were stained with hematoxylin/eosin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171799&req=5

fig1: Keratinocyte-specific disruption of c-raf-1 by the Cre-loxP system. (A) PCR genotyping of keratinocytes (KC) and tail genomic DNA preparations (T). M, marker. (B) Lack of Raf-1 protein in c-raf-1Δ/Δep keratinocytes. Keratinocytes isolated from six independent animals were lysed and subjected to Western blotting with an α-Raf kinase domain mAb, which recognizes all three Raf kinases. The genotype of the mice is indicated at the bottom of the panel. The band corresponding to Raf-1 is absent in K5-Cre+ cultures. (C) c-raf-1Δ/Δep mice display waved fur during the first hair cycle. A 4-wk old f/f and a c-raf-1Δ/Δep littermate are shown. (D) Normal epidermal architecture in c-raf-1Δ/Δep newborn mice. Paraffin sections from f/f or c-raf-1Δ/Δep skin were stained with hematoxylin/eosin.
Mentions: We have used a Cre transgene expressed in the basal epidermal layer and follicular keratinocytes (Tarutani et al., 1997), to induce epidermis-specific ablation of the c-raf-1 gene. Primary keratinocytes derived from mice carrying an homozygous c-raf-1flox/flox (f/f) allele (Jesenberger et al., 2001) and the K5Cre transgene (c-raf-1Δ/Δep, deleted in epidermis) showed complete conversion of the c-raf-1flox to the c-raf-1Δ allele by PCR and lacked Raf-1 protein (Fig. 1, A and B). c-raf-1Δ/Δep mice were viable, fertile, and healthy. At 4 wk old, they displayed curled whiskers and a wavy fur (Fig. 1 C), a phenotype lost after the first hair cycle. The architecture of the epidermis (Fig. 1 D) as well as the expression of basal keratin and differentiation markers in the epidermis and hair follicles was indistinguishable in c-raf-1Δ/Δep mice and control littermates (not depicted). Thus, Raf-1 is not essential in epidermal development and homeostasis. Raf-1 ablation, however, markedly affected wound healing. Control mice closed and reepithelialized full thickness wounds (6-mm ∅) by day 9 after wounding, whereas >40% of the wound was still open in c-raf-1Δ/Δep mice at this time (not depicted). The wounds were not yet closed on day 12, when control mice had already completed both reepithelialization and clearance of cell debris underneath the wound crust (Fig. 2 A). c-raf-1Δ/Δep mice healed the wounds 4–5 d later, and the healed skin was indistinguishable from that of f/f mice (not depicted). The secondary responses of dermal components (granulation, inflammation, and neo-vascularization) were not affected by Raf-1 ablation in keratinocytes. The number of proliferating keratinocytes around the wound site was similarly unaffected (31 ± 10.8 Ki67+ cells in f/f, 32.5 ± 8.8 in Δ/Δep), and TUNEL staining of the migrating epithelial sheets did not show any apoptotic cells in c-raf-1Δ/Δep or control wounds (not depicted). Thus, lack of Raf-1 significantly delayed wound healing in the absence of proliferation or survival defects, suggesting that Raf-1 might be needed for keratinocyte migration.

Bottom Line: These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane.Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration.Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Genetics, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

Show MeSH
Related in: MedlinePlus